Statin-like compounds

ABSTRACT

The present invention relates to compounds with structures resembling natural statins isolated from  Penicillium  sp. (Formula I); where R 1  represents OH, C 6 H 5 COO, R 6 COO and R 6  represents C 1 -C 5  alkyl; R 2-4  represents H, C 1 -C 5  alkyl C 1 -C 5  acyl, R 5  represents H, CH 3 ; and X represents a compound of (Formula II) or (Formula III).

FIELD OF INVENTION

The present invention relates to compounds with structures resemblingnatural statins isolated from Penicillium spp. In particular the presentinvention relates to isolated compounds having UV max relativeabsorbance spectra as measured by HPLC-DAD, of 222-224 nm (100 %),229-234 nm shoulder (84-89%), 290 nm shoulder (13%), 303-305 nm(14-15%), 318-320 nm shoulder (9-11%), 331-333 nm (7.5-8.5%). Anotheraspect of the present invention relates to a pharmaceutical compositioncomprising the compounds of the invention and the use of the compoundsas a medicament. Still another aspect of the invention relates to amethod for the production of a compound of the inventention. Also anaspect of the invention relates to a method of reducing the level oflow-density lipoprotein cholesterol in a human or animal. A furtheraspect relates to a method of producing a compound of the invention. Theisolated compounds can be used in therapy for treatment ofhypercholesterolemia or osteoporosis or as precursors for lead compoundswith therapeutic effect.

BACKGROUND OF THE INVENTION

The statins comprise natural products (polyketides) from fungi that havea series of strong effects on some of the most important human diseases.

Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme Areductase (HMGR). HMGR is the enzyme responsible for catalyzing theconversion of HMG-CoA to mevalonate, which is an early and rate-limitingstep in the cholesterol biosynthetic pathway. Statins are effectivelipid lowering agents and thus have been widely used for lowering serumcholesterol levels in the treatment of hypercholesterolemia reducing therisk of heart attacks. Statins also promote bone formation by affectingthe expression of the bone morphogenic protein-2 (BMP-2) and thisstimulation can have beneficial effects for the treatment of bonefractures and osteoporosis (Mundy et al., Science(1999) 286: 1946-1949;Sugiyama et al., Biochem Biophys Res Commun (2000) 271:688-92). Recentlystatins have recently also been recognized as a new type ofimmunomodulator by acting as direct inhibitors of induction of MHC-IIexpression by INF-γ and thus as repressors of MHC-II-mediated T-cellactivation (Kwark et al., Nature Med., (2001) 6: 1399-1402). Naturallyoccurring statins have antifungal activity (Auclair et al., Bioorganicand Medicinal Chemistry Letters (2001), 11: 1527-1531). Chemicallymodified statin molecules vary in their retention time in the body, thearea of accumulation in the body and effectiveness per dose among otherdifferences (Motti et al., Ann Ital Med Int, 2000, 15: 96-102).

The naturally occurring statins can be subdivided into mevinolins(=monacolins) and compactins. The mevinolins are produced by Aspergillusterreus and Monascus ruber, while Endo et al. (Journal Antibiotics,(1976), 29: 1346-1348) reports the production of compactins byPenicillium citrinum.

Istvan and Deisenhofer, (Science (2001) 292: 1160-1164) have recentlydisclosed the presumed mechanism of inhibition of the HMGR by naturaland chemically synthesized statins.

Due to pleiotropic effects of statins, their reported side effects(gastrointestinal disorders, skin rashes and headache (U.S. Pat. No.6,245,800)), and the variation in retention time in the body, andeffectiveness per dose, a need still exists for statin molecules withunique and desirable properties such as increased effectiveness, lesstoxicity, altered retention time or concentration in a particular organcompared to commercially available statins.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to isolated compoundshaving UV max relative absorbance spectra as measured by HPLC-DAD, of222-224 nm (100%), 229-234 nm shoulder (84-89%), 290 nm shoulder (13%),303-305 nm (14-15%), 318-320 nm should (9-11%), 331-333 nm (7.5-8.5%),and UV min relative absorbance spectra as measured by HPLC-DAD at202-204 nm (26-45%), 254-258 nm (2-3%), 326-328 nm (6-8%).

In a second aspect the present invention relates to a compound offormula (IV),

where X represents a compound of formula (II) or (III),

In a third aspect the present invention relates to a pharmaceuticalcomposition comprising at least one of the compounds(s) according to theinvention or solistatin or a pharmaceutically acceptable salt ormetabolite or prodrug thereof, and a pharmaceutical acceptable carrier.

In a fourth aspect the present invention relates to a use of thecompound(s) according to the invention or solistatin for the productionof a medicament.

In a fifth aspect the present invention relates to a use of thecompound(s) of the invention or solistatin as a medicament.

In a sixth aspect the present invention relates to a use of thecompound(s) of the invention for the manufacture of a medicament for thetreatment of hypercholesterolemia and/or osteoporosis, or for use as animmunosuppessor.

In a seventh aspect the present invention relates to a method ofreducing the level of low-density lipoprotein cholesterol (LDL-C) in ahuman or animal by administering an effective amount of at least onecompound according to the invention or solistatin. In an eigths aspectthe present invention relates to a method of producing a compound of theinvention, which method comprises

-   a) culturing a organism in a suitable culture medium under    conditions promoting the production of the said compound(s); and-   b) recovering the compound(s) from the organism or culture medium    obtained in step (a).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows UV-absorbance spectra as measured by HPLC-DAD for theStatans A, A₁, and B.

FIG. 2 shows the development in total plasma cholesteol (avarege) ofStatan A in rabbits.

DEFINITIONS

Prior to a discussion of the detailed embodiments of the invention, adefinition of specific terms related to the main aspects of theinvention is provided.

“Statin”: The term “statin” according to the present invention means anyinhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR),wherein a HMG-like moiety (present in all known statins) of the statincompetes for binding at the active site of the HMG-reductase, with theHMG-moiety of HMG-CoA.

“Solistatin” is a compound of formula (I) , wherein R₁ is H; R₂₋₄ is H;R₅ is H, and X is a compound of formula (II).

“Retention index”: The retention index or bracketed retention index isbased on a series of alkylphenone standards (acetophenone,propiophenone, butyrophenone, valerophenone, hexanophenone,octanophenone and decanophenone) run before each series of HPLC runs.Acetophenone has the RI value 800 (because there are 8 carbon atoms inthe molecule), propiophenone is 900 (9 carbon atoms) etc. Based on theretention time of a compound from an extract, the compound is placedbetween the two alkylphenones eluting before and after the saidcompound, and the RI value is calculated by linear interpolation (seeformula in Frisvad and Thrane, J. Chromatogr.(1987) 404:195-214).

“HPLC-DAD”: High performance liquid chromatography with diode arraydetection (UV detector). UV absorbance of the compound is measured atdifferent (a spectrum) wavelengths simultaneously.

DETAILED DESCRIPTION OF THE INVENTION

The mevinolins only include one or two double-bonds (the latter atransoid conjugated diene) in the naphthyl part of the molecules givingeither uncharacteristic end-absorbtions or the characteristic UVspectrum of the main 1,2,6,7,8,8a-hexahydronaphthyl group (absorbtionsat 231, 238 and 247 nm, see FIG. 1 in Endo, J. Antibiotics (1979),32:852-854). This characteristic chromophore has only been found in thenaturally occurring statins and in versiol (LL-N313epsilon). The latterhas been found in Aspergillus versicolor and Sporormia affinis (Turner,Fungal metabolites, Academic Press, London (1971); Turner and Aldridge,Fungal metabolites II, Academic Press, London(1983)). Versiol does nothave the delta-lactone (3-hydroxyheptan-5-olide) or its active openacidic form that is important for the activity of the statins (Istvanand Deisenhofer, Science (2001) 292: 1160-1164). For this reason one canscreen for statins using high performance liquid chromatography withdiode array detection (UV detection) (=HPLC-DAD) (Frisvad and Thrane, J.Chromatogr.(1987) 404:195-214; Frisvad and Thrane, In Betina, V. (Ed.)Chromatography of mycotoxins: techniques and applications J. ofChromatography Library (1993) 54: 253-372) in order to recognize thecharacteristic chromophore of the 1,2,6,7,8,8a-hexahydronaphthyl groupas long as versiol can be excluded. This can be verified by usingexternal standards of mevinolin and compactin in the chromatographicanalyses. Mevinolin is always the main statin in the mevinolin secondarymetabolite biosynthetic family and compactin is always the main statinin the compactin secondary metabolite biosynthetic family and at leastone of them should be present in an extract in order to claim thatstatins are produced by an organism.

Using the above method fresh isolates of Penicillium solitum, wereidentified as comprising compounds (herein called statans) comprising achromophore different both from the characteristic chromophore of the1,2,6,8,8a-hexahydronaphthyl group present in both compactin andmevinolin and the naphthyl group of solistatin as evidenced byUV-spectral data. Said chromophore is different from known secondarymetabolites, but has a resemblance to the chromophore of8-methoxy-1-naphthol (see Stadler, M. et al., Mycological Research105(10): 1191-1205) and the presence of which is consistently linked toa few compactin producing species. The said chromophore comprised in thecompounds of the invention (the statans) and isolated from statinproducing isolates of P. solitum can be characterized by their uniqueUV-spectra.

In a first aspect the present invention therefore relates to isolatedcompounds having UV max relative absorbance spectra as measured byHPLC-DAD, of 222-224 nm (100%), 229-234 nm shoulder (84-89%), 290 nmshoulder (13%), 303-305 nm (14-15%), 318-320 nm shoulder (9-11%),331-333 nm (7.5-8.5%), and UV min relative absorbance spectra asmeasured by HPLC-DAD at 202-204 nm (26-45%), 254-258 nm (2-3%), 326-328nm (6-8%). The isolated compounds characterized by the above UV-spectra(also shown in FIG. 1) are only found in a few compactin producingfungus species, especially of the genus Penicillium. The isolatedcompounds according to the present invention should according to theobserved spectra above have structures resembling the naturallyoccurring statins known as compactin (also known as mevastatin),simvastatin, lovastatin and pravastatin. The compounds could be newmembers of the statin family or they could be intermediates in thestatin metabolic pathway.

In one embodiment of the present invention the isolated compoundscomprise statins.

The isolated compounds have similar UV-spectra as defined above, andtheir alkyl phenone retention index (RI), as defined earlier, iscomprised in the range from 887-967 as shown in the example. Furthermorethe isolated compounds (statan B, statan A, and statan A₁) can becharacterized by having a RI selected from the ranges 891-904, 946-959,or 960-964. The compounds according to the present invention are alsohighly fluorescent when using an excitation wavelength of 230 nm and anemission wavelength of 450 nm.

NMR-Spectrum

As described in the examples, the compound of the invention was purifiedand NMR-spectra were recorded at 600.13 MHz for ¹H and 150.92 MHz for¹³C. The chemical shifts are given relative to DMSO (DimethylSulfoxide), 2.5 ppm for ¹H and 39.5 ppm for ¹³C. The NMR-data resultedin a chemical structure of formula (IV) as shown below.

wherein X represents a compound of formula (II) or (III)

Statan A is the compound of formula (IV) wherein X is the group shown informula (II). Statan A may advantageously be used according to theinvention as intermediate for preparing other statans of the inventionas depicted in formula (I).

In a further aspect the present invention relates to compounds offormula (I)

where R₁ represents OH, C₆H₅COO, R₆COO and R₆ represents C₁-C₅ alkyl;

-   R₂₋₄ represents H, C₁-C₅ alkyl, or C₁-C₅ acyl-   R₅ represents H, CH₃; and-   X represents a compound of formula (II) or (III)-   R₆ can be a branched or straight alkyl.    The compounds of formula (I) can be produced from the compound of    formula (IV).

Compounds of formula (I) is in context of the present invention referredto as “statans”. R₂, R₃, R₄ can be substituted with C₁-C₅ alkyl or C₁-C₅acyl by a Fridel Kraft alkylation or acylation as described in example8. R₁ representing R₆COO where R₆ represents C₁-C₅ alkyl can be producedfrom a compound of formula (IV) by esterification of the R₁ position asdescribed in examples 5 and 6.

In an embodiment R₁ is 2-methylbuturyl or 2-dimethylbutyryl or C₆H₅COO(or PhCOO). Benzylation of Statan A at R₁ is described in example 7.

Alternatively the esterification may be carried out by a processemploying an immobilized lipase enzyme in a nonaqueous system. Anexample of such a lipase enzyme is Candida cylindracea C (Type VII fromSigma Chemical Co.). The process is described in U.S. Pat. No. 5,420,024and furthermore lists microbes which make the enzyme with thecorresponding deposit numbers (see table 1 in U.S. Pat. No. 5,420,024),the content of which is incorporated by reference. Compounds which maybe prepared include those wherein R is R₆ is ethyl, n-propyl, 2-butyl or2-methyl-2-butyl.

Statan A can in a further embodiment be used for ether formation, e.g.the formation of statan butyl ether as described in example 9.

R₅ can be substituted with methyl using any method know in the art,e.g., as described in TETRAB, Tetrahedron, 44, 18, 1988: 5745-5760 or J.Org. Chem, 48, 17, 1983: 2814-2820 or shown in examples 11 and 12.

Within its scope the invention includes all optical isomers of compoundsof the present invention, some of which are optically active, and alsotheir mixtures including racemic mixtures thereof.

Within the scope of the invention are all tautomeric forms of thecompounds of the present invention as well as metabolites or prodrugs.

The compounds of the present invention are found in compactin producingspecies. Accordingly in one embodiment of the present invention theisolated compounds described above are derived from Penicillium species,including preferably psychrophilic or psychrotolerant species ofPenicillium. In another embodiment the said Penicillium sp is P.solitum. A suitable P.solitum strain for providing the compounds of thepresent invention could in a further embodiment be P.solitum CBS 147.86(deposited at Centraalbureau voor Schimmelcultures). Also the belowdeposited Penicillium species are specifically contemplated, i.e., P.neocommune, preferably strain CBS 111,239; P. novoniwotense, preferablystrain CBS 111,240; P. landeri, preferably strain CBS 111,241; P.lanosum, preferably strain CBS 111,243; and P. pinicola, preferably CBS111,244.

As discussed above statins have been demonstrated to have severalbeneficial therapeutic effects as e.g. lipid lowering agents used forlowering serum cholesterol levels, promotion of bone formation for usein osteoporosis, immunosuppessing effects, and new and improvedpleiotropic effect.

A further aspect of the present invention therefore relates to apharmaceutical composition comprising at least one compound according tothe invention, or a pharmaceutically acceptable salt or metabolite orprodrug thereof, and a pharmaceutical acceptable carrier.

A “metabolite” of a compound disclosed herein is an active derivative ofa compound disclosed herein which is produced when the compound ismetabolized. Metabolites of compounds disclosed herein can be identifiedeither by administration of a compound to a host and an analysis ofblood samples from the host, or by incubation of compounds with hepaticcells in vitro and analysis of the incubant.

A “prodrug” is a compound that either is converted into a compounddisclosed in the application in vivo or has the same active metaboliteas a compound disclosed in this application.

Such prodrug form could e.g. be a physiologically-hydrolysable andacceptable ester meaning an ester in which the hydroxyl group isesterified and which is hydrolysable under physiological conditions toyield an acid which is itself physiologically tolerable, at dosages tobe administered. The term is thus to be understood as defining regularprodrug forms. Examples of such esters include for example acetates, aswell as benzoates of the compounds of the invention.

The salts include pharmaceutically acceptable acid addition salts,pharmaceutically acceptable metal salts or optionally alkylated ammoniumsalts, such as hydrochloric, hydrobromic, hydroiodic, phosphoric,sulfuric, trifluoroacetic, trichloroacetic, oxalic, maleic, pyruvic,malonic, succinic, citric, tartaric, fumaric, mandelic, benzoic,cinnamic, methane-sulfonic, ethane sulfonic, picric and the like, andindude acids related to the pharmaceutically acceptable salts listed inJournal of Pharmaceutical Science, 66, 2 (1977) and incorporated hereinby reference, or lithium, sodium, potassium, magnesium and the like.Furthermore, the pharmaceutical composition of the invention maycomprise at least one compound according to the invention combined withone or more other pharmacologically active compounds.

In another embodiment of the invention the pharmaceutical compositionfurther comprises acetyl salicylic acid.

In a still further embodiment the invention relates to a method for theproduction of a compound of formula (V) or (I). Formula (V) is formula(I), where R₁ represents OH, R₂₋₄ represents H, R₅ represents CH₃, and Xrepresents a compound of formula (II) or (III).

Compounds of formula (V) may be prepared by methylation of position R₅of the compound of formula (IV) of the invention. Methylation may becarried out using any method known in the art, for instance, asdescribed above or in examples 11 or 12.

Pharmaceutical Compositions

The present invention also relates to pharmaceutical compositionscomprising, as an active ingredient, at least one of the compounds ofthe present invention or a pharmaceutically acceptable salt thereof and,usually, such compositions also contain a pharmaceutically acceptablecarrier or diluent.

Pharmaceutical compositions comprising a compound of the presentinvention may be prepared by conventional techniques, e.g. as describedin Remington: The Science and Practise of Pharmacy, 19^(th) Ed., 1995.The compositions may appear in conventional forms, for example capsules,tablets, aerosols, solutions or suspensions.

Typical compositions include a compound of the present invention or apharmaceutically acceptable acid addition salt thereof, associated witha pharmaceutically acceptable excipient which may be a carrier or adiluent or be diluted by a carrier, or enclosed within a carrier whichcan be in form of a capsule, sachet, paper or other container. In makingthe compositions, conventional techniques for the preparation ofpharmaceutical compositions may be used. For example, the activecompound will usually be mixed with a carrier, or diluted by a carrier,or enclosed within a carrier, which may be in the form of an ampoule,capsule, sachet, paper, or other container. When the carrier serves as adiluent, it may be solid, semi-solid, or liquid material, which acts asa vehicle, excipient, or medium for the active compound. The activecompound can be adsorbed on a granular solid container for example in asachet. Some examples of suitable carriers are water, salt solutions,alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil,syrup, peanut oil, olive oil, gelatine, lactose, terra alba, sucrose,cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin,acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid,fatty acids, fatty acid amines, fatty acid monoglycerides anddiglycerides, pentaerythritol fatty acid esters, polyoxyethylene,hydroxymethylcellulose and polyvinylpyrrolidone.

The formulations may also include wetting agents, emulsifying andsuspending agents, preserving agents, sweetening agents or flavouringagents.

The pharmaceutical preparations can be sterilized and mixed, if desired,with auxiliary agents, emulsifiers, salt for influencing osmoticpressure, buffers and/or coloring substances and the like, which do notdeleteriously react with the active compounds.

The route of administration may be any route, which effectivelytransports the active compound to the appropriate or desired site ofaction, such as oral, nasal, pulmonary, transdermal or parenteral e.g.rectal, depot, subcutaneous, intramuscular or intranasal, the oral routebeing preferred.

If a solid carrier is used for oral administration, the preparation maybe tabletted, placed in a hard gelatin capsule in powder or pellet formor it can be in the form of a troche or lozenge. If a liquid carrier isused, the preparation may be in the form of a syrup, emulsion, softgelatin capsule or sterile injectable liquid such as an aqueous ornon-aqueous liquid suspension or solution.

For nasal administration, the preparation may contain a compound of thepresent invention dissolved or suspended in a liquid carrier, inparticular an aqueous carrier, for aerosol application. The carrier maycontain additives such as solubilizing agents, e.g. propylene glycol,surfactants, absorption enhancers such as lecithin (phosphatidylcholine)or cyclodextrin, or preservatives such as parabenes.

Tablets, dragees, or capsules having talc and/or a carbohydrate carrieror binder or the like are particularly suitable for oral application.Preferable carriers for tablets, dragees, or capsules include lactose,corn starch, and/or potato starch. A syrup or elixir can be used incases where a sweetened vehicle can be employed.

The compounds of the invention may be administered to a mammal,especially a human, in need of such reducing or lowering of the intakeof fat food. Such mammals include also animals, both domestic animals,e.g. household pets, and non-domestic animals such as wildlife.

The compounds of the invention may be administered in the form of analkali metal or earth alkali metal salt thereof, concurrently,simultaneously, or together with a pharmaceutically acceptable carrieror diluent, especially and preferably in the form of a pharmaceuticalcomposition thereof, in an effective amount.

Pharmaceutical compositions containing a compound according to theinvention may be administered one or more times per day or week,conveniently administered at mealtimes. An effective amount of such apharmaceutical composition is the amount that provides a clinicallysignificant effect against consumption of fat food. Such amounts willdepend, in part, on the particular condition to be treated, age, weight,and general health of the patient, and other factors evident to thoseskilled in the art.

The structure of the compounds according to the invention resembles thestructure of naturally occurring statins and a further embodiment of theinvention thus relates to the use of the said compounds for theproduction of a statin. The said statin comprises in another embodimentcompactin, simvastatin, lovastatin, and pravastatin.

In another embodiment the present invention relates to a use of thecompounds of the invention as a medicament. In a still furtherembodiment the present invention relates to a use of the compounds ofthe invention for the manufacture of a medicament for the treatment ofhypercholesterolemia or osteoporosis or as an immunosuppressor.

A further aspect of the present invention relates to a method ofreducing the level of low-density lipoprotein cholesterol (LDL-C) in ahuman or animal by administering an effective amount of at least onecompound according to the invention. In a further embodiment theinvention relates to a use of solstatin (Sørensen et al., 1999,Phytochemistry 51:1027-1029) for the preparation of a medicament and ina particular embodiment for the use of the said medicament for treatmentof hypercholesterolemia or osteoporosis or as an immunosuppressor.

The compounds of the invention can be produced in any conventional wayknown to the skilled person by providing a suitable host organismcomprising the compound of the invention and growing the said organismin a suitable culture medium under conditions promoting the productionof the said compound(s) and recovering the compound(s) from the hostorganism or culture medium.

It is also contemplated that the culture broth comprising statans aftergrowing the organisms of the invention can be use for the preparation ofa pharmaceutical composition.

The said host organism is in one embodiment a fungus.

In another embodiment the said host organism is a Penicillium sp. likee.g. a P. solitum, including P. solitum CBS 147.86 (redeposited asPenicillium solitum CBS 111.242 (NN049656) on 29 Nov. 2002) or one ofthe following deposited strains: Deposit NZ Number Accession Number Dateof Deposit Penicillium sp. NN049653 CBS 111239 29 Nov. 2002 Penicilliumsp. NN049654 CBS 111240 29 Nov. 2002 Penicillium sp. NN049655 CBS 11124129 Nov. 2002 Penicillium sp. NN049657 CBS 111243 29 Nov. 2002Penicillium sp. NN049658 CBS 111244 29 Nov. 2002

The strains have been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication.

These strains were deposited on under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure at Centraalbureau voor Schimmelcultures(CBS), Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.

Strain CBS 111,239 (NN 049653) may be referred to as Penicilliumneocommune Frisvad (sp.nov) and originates from Wyoming National Forest,US.

Strain CBS 111,240 (NN 049654) may be referred to as Penicilliumnovoniwotense Frisvad (sp.nov) and originates from Faeroe Islands,Denmark.

Strain CBS 111,241 (NN 049655) may be refer to as Penicillium landeriFrisvad (sp.nov) and originates from Wind River Range, Wyo., US.

Strain CBS 111,242 (NN049656) is of the species Penicillium solitum andhas been isolated from apple, Denmark.

Strain CBS 111,243 (NN049657) is of the species Penicillium lanosum andhas been isolated from soil under spruce tree, Brandy Wine Falls,British Colombia, Canada.

Strain CBS 111,244 (NN049658) may be referred to a Penicillium pinicolaFrisvad (sp.nov) and originates from Wyom., US.

Statin Activity

Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme Areductase (HMGR). HMGR is the enzyme responsible for catalyzing theconversion of HMG-CoA to mevalonate, which is an early and rate-limitingstep in the cholesterol biosynthetic pathway. The compounds of thepresent invention can be analyzed for their effectiveness as HMGRinhibitors by any method that can directly or indirectly measure thelevel of LDL-cholesterol in the subject. One such test could be theCOBAS INTEGRA® HDL-Cholesterol Direct (Roche Diagnostics). Nielsen etal. (Pharmacology & Toxicology, 1993, 72: 148-151) and Holm et al.(Arteriosclerosis, Thrombosis, and Vascular Biology, 1997, 17:2264-2272) describes methods for the determination of various forms ofcholesterol, both references are hereby incorporated by reference.

In the examples characterization of two distinct compounds is describedas well as NMR-spectral analysis for the isolated compound of formula(IV), and animal studies showing the HMGR inhibiting effect of thecompounds of the invention.

EXAMPLES Example 1

Screening for Statans

The fungus Penicillium solitum CBS 147.86 (redeposited as CBS 111.242)is inoculated from a resuspension of a preserved freeze-dried spore andinoculated on malt extract agar [according to Blakeslee] (See Samson, R.A., Hoekstra, E. S., Frisvad, J. C. and Filtenborg, O. (eds.)Introduction to food- and airborne fungi. 6th edition., 2000, 378-382)and incubated at 20° C. for 1 week. The conidia are suspended in peptone(0.1%) in water to produce a suspension of approximately 10.000 conidiapr ml. From this suspension 11 cm Petri dishes with 17 ml YES (yeastextract sucrose) agar (Samson, R. A., Hoekstra, E. S., Frisvad, J. C.and Filtenborg, O. (eds.) Introduction to food- and airborne fungi. 6thedition., 2000, 378-382) are 3-point inoculated with a inoculationneedle and the YES agar plates incubated at 25° C. for two weeks in thedark. The content of 10 Petri dishes is extracted in a ColworthStomacher 400 for 2 min with 200 ml ethyl acetate with 1% formic acid.The organic phase is filtered using a hydrophobic filter and the organicsolvents evaporated in a rotary evaporator under vacuum at 40° C. Theremaining dry extract is re-dissolved in ½ ml methanol, filtered andinjected into the HPLC-DAD system (Agilent). The HPLC-DAD system shouldbe connected with an effective fluorescence detector in order to confirmthe presence of statans. All compounds should be analyzed using theHPLC-DAD system of Frisvad and Thrane (Frisvad and Thrane, J.Chromatogr.(1987) 404:195-214; Frisvad and Thrane In Betina, V.(Ed.)Chromatography of mocotoxins: techniques and applications J. ofChromatography Library (1993) 54: 253-372) as modified by Smedsgaard (J.Chromatogr. A ,1997, 760: 264-270) with compactin as an external andinternal standard and alkylphenones as external standards. In thatsystem the new statin-like compounds (statans) have the followingproperties:

Statans: Statan A: RI (alkylphenone retention index) average 953 (range946-959), Statan A₁: RI average 961 (range 960-964), statan B: RIaverage 898 (range 891-904) (as compared to compactin RI average 1108).UV spectra as measured by DAD: (UV max, relative abs.): 222-224 nm(100%), 229-234 nm shoulder (84-89%), 290 nm shoulder (13%) 303-305 nm(14-15%), 318-320 nm shoulder (9-11%), 331-333 nm (7.5-8.5%) (abs.minima at 202-204 nm (2645%), 254-258 nm (2-3%), 326-328 nm (6-8%)). Thestatans are highly fluorescing when excitation is performed at 230 nmand using an emission wavelength of 450 nm. Few other secondarymetabolites from fungi fluoresce at these conditions (an example isochratoxin A and B, but these have completely different UV spectra andretention indices).

Example 2

Production and Isolation of Statan A

The isolate CBS 147.86 (redeposited as CBS 111.242) was cultured for 2weeks in the dark at 20° C. as three point mass inoculations on 1000Petri dishes (9 cm) on about 20 liter of yeast extract sucrose agar(YES) (yeast extract 20 gr, sucrose 150 g, agar 20 g, distilled water).

The content of the Petri dishes were extracted twice with 10 liter ofEtOAC to give ca. 25 9 of crude extract which was partitioned between atotal of 1 liter of heptane and 1 liter of 90% MeOH in order to removelipids and apolar compounds. The MeOH fraction was diluted to 60% andextracted with a total of 1 liter of CH₂Cl₂ to give a total of 8 g ofStatan A (formula IV) rich extract. This extract was then coated onto 8g of Celite and divided into three portions which subsequently wereseparated by vacuum liquid chromatography on a C₁₈ SPE column (10 g)into four fractions using 100 ml of H₂O/MeOH in the following amountsA:(75:25), B:(50:50), C:(25:75) and D:(0:100). The C fraction (4.6 g)rich in Statan A was further separated on a Sephadex LH20 column (25×900mm) using MeOH at 1 mL/min to give 8 fractions (CA-CF). Finally the CEfraction (96 mg) was subjected to HPLC separation on a Waters PrepNova-Pak C18 cartridge (25×100 mm, 6 micro m) using H₂O/CH₃CN (65:35) asmobile phase to give 30 mg of pure Statan A.

Example 3

Production of Statans in Submerged Culture Preparation of InoculationMaterial

A petri dish with PDA (potato dextrose agar) was inoculated (at3-points) with fungal isolate P. solitum CBS 147.86 (redeposited as CBS111.242) and incubated at 25° C. for 7 days.

Conidia were harvested by addition of 5 mL sterile H₂O, holding 0.1%Tween per plate. The conidia were released from the mycelium by gentlescraping with pipette tip. The resulting suspension of conidia werepoooled from two plates at a time and added to a flask with a totalvolume of 100 mL H₂O (0.1% Tween, final concentration).

Culture Conditions

A fed batch culture condition in 2L scale was used.

-   Composition of medium:-   20 g/L yeast extract (Difco),-   20 g/L sucrose,-   18 mg/L ZnSO₄.7H₂O,-   8 mg/L CuSO₄.5H₂O, 1 ml/L Pluronic (antifoam), pH ajusted to 5,0    prior to sterilization.-   Feed: 500 g/L sucrose, 2 ml/L Pluronic (antifoam).-   The fermentation tank was inoculated by adding 2 mL of the spore    suspension (described above).-   Feed start: 36 h after inoculation, feed rate 2.5 g/L/h.-   pH control: >4 (10% NaOH) and <6 (10% H₃PO₄).-   Temperature: 21° C.-   Stirrer speed: start 500 rpm, increased to keep dissolved    oxygen >50%, max 1100 rpm.-   Airflow: 1 L/min.-   Fermentation time 185 h (=8 days).    Extraction of Culture Broth

3 mL of full culture broth was extracted with 3 mL of ethylene acetate(with 1 v/v % formic acid) for 1 hour in ultrasound bath. The ethyleneacetate was transferred to a vial and evaporated in a rotary vacuumconcentrator. 0.5 mL of methanol was used to redissolve the remains for1 hour in ultrasound bath. The methanol was filtered though a 0.22 microm filter into a HPLC-vial.

-   HPLC conditions: Column: Agilent, Hypersil with BDS-C18, 3 um    (mymeter), 4.0×100 mm;-   Injection: 10 microL-   Flow: 1 mL/min-   Gradient: Start at 85% MilliQ H₂O (with 50 ppm TFA) and 15%    acetonitrile (with 50 ppm TFA) until 40 min where 100% acetonitrile    is reached. 40 to 45 minutes 100% acetonitrile.    Results

The fermentation gave good levels of several statans. Further thesefermentation conditions gave a surprisingly low level of othermetabolites.

Example 4

Structural Elucidation of Statan A

Accurate mass analysis was done on a Agilent HP 1100 liguidchromatograph with a diode array detector (DAD) coupled to a LCTMicromass oaTOF instrument with Z-spray electro spray source (ESI) and alockspray probe. 1 microL of sample was injected on an Agilent HypersilBDS-C₁₈ 125×2 mm column with 3 micro m particles. A water-acetonitrilegradient, starting with 15% acetonitrile-water going to 100%acetonitrile in 40 min, maintaining 100% acetonitrile for 5 min, beforereturning to the start conditions in 8 min equilibrating for 5 min. TFA,50 ppm was added to the water. The MS was operated in the positive ESImode using leucineenkephalin as lockmass ([M+H]+ion at 556.2771 Da/e).The molecular ion of protonated Statan A appeared at 301.1421corresponding to the composition C₁₈H₂₀O₄ (Δ−6.3 ppm) corresponding to 9DBE.

NMR spectra of statans were recorded in 5 mm tubes at 600.13 MHz for ¹Hand at 150.92 MHz for ¹³C and at 300 K, using DMSO-d₆, on a Bruker DRX600 according to Larsen et al., 2001, (J Agricult and Food Chem 49:5081-5084). The ¹³C and the C,H-COSY spectra revealed the presence ofone methyl, four methylene, and two methine groups, together with 5aromatic protons, two hydroxy protons (9.95 and 5.20 ppm), 5 quaternaryaromatic carbon atoms and finally a carboxy group at 170.2 indicating anester or lactone. The ¹H and H,H-COSY spectra revealed the presence ofone aliphatic (—CH₂—CHOH—CH₂—CH—CH₂—CH₂—) and two aromatic spin systems(AB and ABC) together with a single methyl group. Interpretation of theheteronuclear multiple bond coherence spectrum (HMBC) established thestructure of Statan A. The chemical shift values of the carbons in thenaphthalene part of the molecule were very similar to the values of themodel compound 7,8-dimethyl-1-naphtol (Jung, K.-Y., Koreeda, M., 1989, JOrganic Chem 54: 5667-5675) and similarly the chemical shift values ofthe aliphatic part of the molecule were very similar to those reportedfor solistatin (Sørensen et al., 1999, Phytochemistry 51: 1027-1029) andother statins, altogether strongly supporting the proposed structure.

¹H NMR (DMSO-d6): δ 9.95 (1H, bs, 8′—OH), 7.54 (1H, d, J=8.2 Hz, H-4′),7.24 (1H, d, J=8.2 Hz, H-3′), 7.24 (1H, d, J=7.9 Hz, H-5′), 7.16 (1H,dd, J=7.9 and 7.5 Hz, H-6′), 6.83 (1H, d, J=7.5 Hz, H-7′), 5.20 (1H, brs, 3—OH), 4.72 (1H, m, 5-H), 4.16, br s, 3-H), 3.45 (1H, br s, H-7a),3.36 (1H, br s, H-7b), 2.68 (1H, dd, J=17.2 and 4.7 Hz, H-2a), 2.44 (3H,s, Me), 2.43 (1H, d m, 17.2 Hz, H-2b), 1.92 (2H, m, H-6), 1.91 (1H, m,H-4a), 1.79 (1H, ddd, 14.3, 11.7 and 3.3 Hz, H4b).

¹³C NMR (DMSO-d6): δ 170.2 (C-1), 154.8 (C-8′), 135.1 (C-1′), 135.1(C-4′a), 132.1 (C-2′), 129.0 (C-3′), 125.8 (C-4′), 124.7 (C-6′), 123.1(C-8′a), 119.5 (C-5′), 109.9 (C-7′),75.5 (C-5), 61.0 (C-3), 38.5 (C-2),36.5 (C-6), 34.8 (C-4), 27.0 (C-7),19.5 (Me).

Example 5

Acetylation of Statan A in Position R₁ in Sodium Hydroxide Synthesis andRecovery of 8-Statanyl Acetate

Dissolve 19 mg of Statan A in 10 percent sodium hydroxide (40 microL) ina glass vial (2 ml). Add crushed ice (about 5 mg) and acetic anhydride(8.2 mg, 7.6 microL) to the vial and shake it for 5 minutes.Subsequently add 200 microL of dichloromethane and shake gently andleave the two phases to separate. Take away the lower layer ofdichloromethane and statan acetate and shake it with dilute sodiumbicarbonate. Add another 200 microL of dichloromethane to the slightlybasic water phase and wash the organic phase with dilute sodiumbicarbonate. Take the combined organic phases to dryness. Redissolve thesynthesis products in 100 microL of methanol and separate the mixture ofmono- an di-acetates by HPLC separation on a Waters Prep Nova-Pak C18cartridge (25×100 mm, 6 micro m) using H₂O/CH₃CN (55:45) as mobile phaseat a flow rate of 2 mL/min to give 15 mg (66%) of pure 8-acetoxy StatanA and 4 mg of di-acetoxy Statan A.

Example 6

Synthesis and Recovery of 8-Statanyl Isobutyrate

Dissolve 28 mg of Statan A in 1 ml of pyridine in a glass vial (15 mL).Add isobutyric anhydride (25 mg) to the vial and leave it at roomtemperature for 12 hr. Add crushed ice and leave the reaction mixturefor 1 hour. Subsequently add 2 mL of dichloromethane and shake gentlyand leave the two phases to separate. Take away the lower layer ofdichloromethane and Statan A isobutyrate and extract the pyridine-waterphase once more with 1 mL of dichloromethane. Wash the combined organicphases first with dilute hydrochloric acid and subsequently with dilutesodium bicarbonate. Take the combined organic phases to dryness.Redissolve the synthesis products in 100 microL of methanol and separatethe mixture of 8-statanyl isobutyrate and the di-isobutyrate by HPLCseparation on a Waters Prep Nova-Pak C18 cartridge (25×100 mm, 6 microm) using H₂O/CH₃CN (55:45) as mobile phase at a flow rate of 2 ml/min togive 24 mg (69%) of pure 8-statanyl isobutyrate.

Example 7

Benzylation of Statan A in Position R₁

Synthesis of 8-Statanyl Benzoate

Dissolve 30 mg of Statan A in 10 percent sodium hydroxide (200 microL)in a stoppered glass flask. Add benzoyl chloride (22 mg, 18 μl), closethe flask, shake vigorously (15 minutes). Add crushed ice and leave for5 minutes. Subsequently add 200 microL of dichloromethane and shakegently and leave the two phases to separate. Take away the lower layerof dichloromethane and statanyl benzoate and shake it with dilute sodiumbicarbonate. Add another 200 microL of dichloromethane to the waterphase and wash the organic phase with dilute sodium bicarbonate. Takethe combined organic phases to dryness to give 34 mg (84%) of 8-statanylbenzoate.

Example 8

Acylation of Statan A in Positions R₂, R₃ and R₄ Synthesis and Recoveryof 4-, 5- and 7-Acetostatan A by Fries Reaction

Phenolic and naphtolic ketones are produced by the rearrangement oftheir phenolic or naphtolic esters in the presence of anhydrousaluminium chloride. Mix powdered 8-statanyl acetate (36 mg) and powderedanhydrous aluminium chloride (41 mg) in a small flaks fitted with an aircondenser which is closed by a calcium chloride tube. Heat the flask inan oil bath, slowly at first to reach ca. 110° C. in 30 minutes and thenat about 160° C. for 1 hour more. Remove the flask from the oil bath,allow to cool, add crushed ice (300 mg) followed by concentratedhydrochloric acid (12 microL) to decompose aluminium salts. Wash thewater phase with another 200 microL of dichloromethane. Take thecombined organic phases to dryness and redissolve in methanol forseparating the reaction mixture by HPLC on a Waters Prep Nova-Pak C18cartridge (25×100 mm, 6 micro m) using H₂O/CH₃CN (55:45) as mobile phaseat a flow rate of 2 mL/min to give pure 4-, 5- and 7-aceto-statan A.

Example 9

Ether formation of Statan A in Position R₁ Statan A ButylEther—Williamson Synthesis Weigh out 11.5 mg (0.5 mmol) of clean sodiuminto a dry flask provided with a double surface condenser, and add 250microL of ethanol. Add a solution of 150 mg (0.5 mmol) of pure Statan Ain 100 microL of absolute ethanol and shake. During shaking for 15minutes add 133 mg (82.5 microL, 0.72 mmol) of butyl iodide. Boil thesolution gently for three hours before distilling off as much aspossible of the alcohol. Add water (400 microL) and dichloromethane (200microL) to the residue in the flask, separate the organic layer and washit twice with with 200 microL portions of 10 per cent sodium hydroxidesolution, then subsequently with water, dilute sulphuric acid and water:dry with magnesium sulphate.

Example 10

Formation of Statan A Carboxylic Acids Synthesis of 5- and 7-Statan ACarboxylic Acids—Kolbe-Schmidt Reaction

Place a solution containing 109 mg of Statan A, 200 mg of potassiumhydrogen carbonate and 5 mL of water in a flask fitted with a refluxcondenser and gas inlet tube. Heat gently on a steam bath for 4 hours;then reflux vigorously over a flame for 30 minutes while passing a rapidstream of carbon dioxide through the solution. Acidify the solutionwhile still hot by adding 180 microL of concentrated hydrochloric acidat the bottom of the flask using a Pasteur pipette. Allow to cool toroom temperature, chill in an ice bath. Subsequently add 2 ml ofdichloromethane and shake gently and leave the two phases to separate.Take away the lower layer of dichloromethane. Wash the water phase withanother 2 ml of dichloromethane and take the combined organic phases todryness. Redissolve the synthesis products in 500 microL of methanol andseparate the mixture of 5- and 7- carboxylic acid Statan A by HPLCseparation on a Waters Prep Nova-Pak C18 cartridge (25×100 mm, 6 microm) using H₂O/CH₃CN (55:45) as mobile phase at a flow rate of 2 mL/min.

Example 11

Triflouroacetylation of Statan A in Positions 3 and 8′ Synthesis andRecovery of 3.8′-Statanyl-di-triflouracetate

Dissolve 38 mg of Statan A in 10 percent sodium hydroxide (200 microL)in a glass vial (2 ml). Add crushed ice (ca. 50 mg) and triflouraceticanhydride (50 mg) to the vial and shake it for 5 minutes. Subsequentlyadd 200 microL of dichloromethane and shake gently and leave the twophases to separate. Take away the lower layer of dichloromethane and andshake it with dilute sodium bicarbonate. Add another 200 microL ofdichloromethane to the slightly basic water phase and wash the organicphase with dilute sodium bicarbonate. Take the combined organic phasesto dryness to recover 58 mg (93%) of the statan di-triflouroacetate.

Example 12

Formation of 6-Methyl-statan A—Friedel Craft

Dissolve 58 mg of 3,8′-statanyl-di-triflouracetate in methylcyclohexane(4 mL) in a small glass flask fitted with a drying tube (CaCl₂). Addanhydrous aluminium chloride (9 mg). Cool the flask in an ice bath andwhile stirring (with a small rotating magnet) add methyl chloride (11mg) during the following 4 hours, maintaining the temperature in thecooling bath at 0-5° C. Stir the mixture for another hour after additionis complete, remove the drying tube, add crushed ice (2 g) in portions,with stirring, to decompose the intermediate addition compounds. Takeaway the upper organic layer and take it to dryness. Redissolve theresidues in 10 percent sodium hydroxide (2 mL) in a glass vial flask andboil the solution under reflux for an hour to remove thetriflouroacetate groups by hydrolysis. Cool the mixture and add dilutesulphuric acid, with stirring, until the solution is acid to litmus.Leave the solution for 30 minutes for the intramolecular lactonisationto complete. Now extract the product from the water phase into (2×500microL) of freshly distilled diethylether. Evaporate of the diethyletherand redissolve the synthesis products in 500 microL of methanol.Separate the mixture by HPLC on a Waters Prep Nova-Pak C18 cartridge(25×100 mm, 6 micro m) using a gradient of H₂O/CH₃CN as mobile phasegoing from (70:30) to (30:70) in 15 minutes using a flow rate of 2ml/min to give 11 mg (30%) of pure 6-methyl-statan A.

Exampel 13

Test of Cholesterol-lowering Effect of Statans in Rabbits

The cholesterol-lowering effect of Statan A was compared against acontrol in male White rabbits (purchased from Hvidesten, DK). Therabbits were housed in individual stainless steel cages with free accessto water. The rabbits were divided into groups with similar baselinevalues of plasma cholesterol, which groups received:

-   1) 5 mg/day PD498 (n=2)(Statan A),-   2) control group (n=4).

The rabbits were treated for 3 days. All animals were fed daily with 100g of a standard rabbit diet (Altromin, Lage, Germany) containing 1%cholesterol. All test drugs were added to the diet. Remaining food wereweighed.

Blood samples for cholesterol determination were drawn at baseline andat the three following days. Blood samples were taken from the marginalear vein and the cholesterol determination was done the same day byenzymatic calorimetric methods, using a commercially available kit(CHOD-PAP, Roche), on a COBAS MIRA auto-analyzer (Roche, Basle, CH).

The results of the present study are shown in FIG. 2. Total plasmacholesterol levels in the Statan A group were consistently lower, whencompared to the control group. These results show a cholesterol-loweringeffect of Statan A with a strong tendency towards lower plasmacholesterol values.

Chow Preparation

Ten kg 1% cholesterol chow: 100 g cholesterol is dissolved in 500 g cornoil by heating to 40° C. The cholesterol mixture is well-mixed with 9400g standard rabbit chow (Altromin, Hvidesten, DK).

-   35 3.5 kg 1% cholesterol chow with test compound is dissolved in 40    mL corn oil and ultrasonicated for 10 min and then well-mixed with    3.5 kg of the cholesterol-enriched chow. 5 mg/day of Statan A: 30 mg    Statan A is dissolved in 1000 microL ethanol (100%). 30 mL corn oil    is added and heated to 40° C. The mixture is well-mixed with 3.5 kg    of the cholesterol-enriched chow.

As an alternative method to the above disclosed method the rabbits areselected as hyper-, normo- or hyporesponsive to dietary cholesterol. Thehyposensitive rabbits are fed with 1% cholesterol chow 5 days beforeadministration of the test drug. Thereafter the rabbits are fed dailyfor 3 days with the cholesterol-enriched (1%) chow containing the testdrugs.

Example 14

Test of Cholesterol-lowering Effect of Solistatin in Rabbits Thecholesterol-lowering effect of solitstatin was tested in rabbits usingthe same method as disclosed in Example 13. The test indicated thatsolistatin had cholesterol-lowering effect.

1-29. (canceled)
 30. Isolated compounds having UV max relativeabsorbance spectra as measured by HPLC-DAD, of 222-224 nm (100%),229-234 nm shoulder (84-89%), 290 nm shoulder (13%), 303-305 nm(14-15%), 318-320 nm shoulder (9-11%), 331-333 nm (75-(7.5-8.5%), and UVmin relative absorbance spectra as measured by HPLC-DAD at 202-204 nm(26-45%), 254-258 nm (2-3%), 326-328 nm (6-8%).
 31. The isolatedcompounds of claim 30, wherein said compounds comprise compounds whichare capable of inhibiting 3-hydroxy-3-methyl glutaryl coenzyme Areductase, such as statins. 32 The isolated compounds according to claim30, wherein the alkyl phenone retention index, (RI), is comprised in therange from 887-967. 33 The isolated compounds according to claim 32,wherein the said RI is 891-904, 946-959, or 960-964. 34 The isolatedcompounds according to claim 30, wherein said compounds fluoresce whenusing an excitation wavelength of 230 nm and an emission wavelength of450 nm. 35 An isolated compound of formula (I)

where R₁ represents OH, C₆H₅COO, R₆COO and R₆ represents C₁-C₅ alkyl;R₂₋₄ represents H, C₁-C₅ alkyl, C₁-C₅ acyl, R₅ represents H, CH₃; and Xrepresents a compound of formula (II) or (III)

36 An isolated compound of formula (I), wherein R₁ is 2-methylbuturyl or2-dimethylbutyryl, R₂ is H, R₅ represents H or CH₃, and X represents acompound of formula (II) or (III). 37 An isolated compound according toclaim 35, having the formula (IV)

where X represents a compound of formula (II) or (III). 38 The isolatedcompounds of claim 30, wherein said compounds are provided from aPenicillium spp. 39 The isolated compounds according to claim 38,wherein said Penicillium spp. is selected from the group consisting ofPenicillium solitum, P. neocommune, P. novoniwotense, P. landeri, P.lanosum, and P. pinicola. 40 The isolated compounds according to claim39, wherein said Penicillium is P. solitum CBS 147.86 (redeposited asCBS 111.242). 41 A pharmaceutical composition comprising: a) at leastone compound according to claim 30, or a pharmaceutically acceptablesalt or metabolite or prodrug thereof; and b) a pharmaceuticalacceptable carrier. 42 The pharmaceutical composition according to claim41, further comprising acetylsalicylic acid. 43 A pharmaceuticalcomposition comprising: b) at least solistatin, or a pharmaceuticallyacceptable salt or metabolite or prodrug thereof; and b) apharmaceutical acceptable carrier. 44 A method of reducing the level oflow-density lipoprotein cholesterol (LDL-C) in a human or animal byadministering an effective amount of at least one compound according toclaim 30 and/or solistatin. 45 A method of producing a compound of claim30, which method comprises a) culturing a organism in a suitable culturemedium under conditions promoting the production of the saidcompound(s); and b) recovering the compound(s) from the host organism orculture medium obtained in step (a). 46 The method of claim 45, whereinthe organism is a fungus. 47 The method of claim 46, wherein theorganism is selected from the group consisting of Penicillium solitum,P. neocommune, P. novoniwotense, P. landeri, P. lanosum, and P.pinicola. 48 A composition comprising the culture broth from a cultureof a Penicillium spp. according to claim 38 prepared by: a) culturingthe cell in a suitable medium under conditions promoting the productionof statin compounds, b) recovering the culture broth obtained in step(a), c) preparing the composition comprising the culture broth. 49 Thecomposition of claim 48, wherein the statin compound(s) are one or morecompounds of claim 1.